Fig. 4. Inhibition of ID4 prevents the effects of BMP4 on glial lineage commitment. The effectiveness of inhibition of Id4 expression by RNAi was confirmed by RT-PCR (A) and quantitative RT-PCR (B) using mRNA extracted from E17 progenitor cells treated with BMP4 (20 ng/ml) for 12 hours and infected with lentivirus containing empty vector (green in B) or Id4-RNAi constructs (blue in B). The graphs shown are representative of experiments done in duplicate three times. (C) E17 progenitor cells infected with lentivirus containing empty vector (parts a-f) or Id4-RNAi (parts g-l) were grown with (parts d-f,j-l) or without (parts a-c,g-i) BMP4 (20 ng/ml) for 2 days. Cells were stained for CNPase, MBP and GFAP. DAPI was used as a nuclear stain and virus-infected cells were identified by staining for GFP. (D) Quantification of CNPase⁺, GFAP⁺ and ß-tubulinIII⁺ cells shows increased numbers of OLs with Id4-RNAi. Scale bars represent means±s.e.m. of four independent experiments. ANOVA was done to determine significance (P<0.05). The control (vector alone) group differed significantly from both BMP4-treated and Id4-RNAi groups with respect to CNPase and GFAP, but did not differ from the group treated with both BMP4 and Id4-RNAi. The BMP4-treated and Id4-RNAi-treated groups each differed from all other groups with respect to CNPase and GFAP. There were no significant differences among groups with respect to ß-tubulinIII.